Chassé H et al. (2016), Cyclin B Translation Depends on mTOR Activity a...

ECB-ART-44557

PLoS One 2016 Mar 04;113:e0150318. doi: 10.1371/journal.pone.0150318.

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Cyclin B Translation Depends on mTOR Activity after Fertilization in Sea Urchin Embryos.

Chassé H , Mulner-Lorillon O , Boulben S , Glippa V , Morales J , Cormier P .

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The cyclin B/CDK1 complex is a key regulator of mitotic entry. Using PP242, a specific ATP-competitive inhibitor of mTOR kinase, we provide evidence that the mTOR signalling pathway controls cyclin B mRNA translation following fertilization in Sphaerechinus granularis and Paracentrotus lividus. We show that PP242 inhibits the degradation of the cap-dependent translation repressor 4E-BP (eukaryotic initiation factor 4E-Binding Protein). PP242 inhibits global protein synthesis, delays cyclin B accumulation, cyclin B/CDK1 complex activation and consequently entry into the mitotic phase of the cell cycle triggered by fertilization. PP242 inhibits cyclin B mRNA recruitment into active polysomes triggered by fertilization. An amount of cyclin B mRNA present in active polysomes appears to be insensitive to PP242 treatment. Taken together, our results suggest that, following sea urchin egg fertilization, cyclin B mRNA translation is controlled by two independent mechanisms: a PP242-sensitive and an additional PP242-insentitive mechanism.

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Species referenced: Echinodermata
Genes referenced: 4e-bp cdk1 LOC100887844 LOC115919910 LOC594261

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	Fig 1. PP242 affects the first mitotic division of S. granularis embryos.(A) Dose-response effect of PP242 on the first mitotic division of sea urchin early development. Batches of S. granularis eggs (5% cells/vol solution in FSW) were pre-incubated 10 min before sperm addition with DMSO (circles) or 3 μM (squares), 10 μM (triangles) or 30 μM (diamonds) PP242. Cleavage rates were scored at different times during the time culture in the continuous presence of the drug. The curve was obtained from the eggs isolated from a single female and was representative of 8 independent experiments. (B) Microscopic observation of nuclear envelope (a-g) and chromatin (a’-g’) morphology in control (a-d and a’-d’) and 10 μM PP242-treated embryos (e-g and e’-g’) at the indicated times post-fertilization.
	Fig 2. PP242 inhibits 4E-BP degradation triggered by fertilization.(A) Total amount of 4E-BP in extracts from control or PP242-treated embryos of S. granularis was analysed by Western blotting using anti-sea urchin 4E-BP antibodies (top panels). CDK1 immunolabelling with PSTAIR antibody was used as a loading control for Western blot (bottom panels). (B) Quantification of the results obtained (in (A)) from control (circles) or PP242 treated (triangles) embryos. 4E-BP amount was normalized against CDK1 level and expressed as a percentage of the value obtained with unfertilized eggs. (C) Quantitation of 4E-BP abundance obtained from untreated (white boxes) and PP242-treated (grey boxes) embryos at the indicated time following fertilization. 4E-BP amount was normalized against CDK1 level and expressed as a percentage of the value obtained with unfertilized eggs. Vertical bars represent Standard Error of the Mean values (SEM) obtained in 6 independent experiments. (*) Indicates a significant difference in 4E-BP amount in PP242-treated embryos in comparison to the untreated embryos (Wilcoxon signed-rank test, p<0.05).
	Fig 3. PP242 inhibits the increase in protein synthesis triggered by fertilization.(A) The rate of in vivo protein synthesis was monitored by the kinetics of [35S]methionine incorporation into proteins. S. granularis eggs (5% cells/vol solution in FSW) were metabolically labelled in the presence of [35S]methionine. After fertilization in the absence (circles) or presence (triangles) of 10 μM PP242, cytosoluble fractions were prepared from 20 μl pelleted embryos and radioactivity incorporation into TCA-precipitated proteins was determined at indicated times. Vertical bars represent Standard Error of the Mean values (SEM) obtained in 3 independent experiments (B) Pattern of proteins translated following fertilization of control (left panel) or PP242-treated (right) embryos. Proteins (30 μg) of the cytosoluble fractions were separated by SDS-PAGE (12%). Radioactive neo-synthetized proteins were visualized by autoradiography. Arrow shows subtle differences in the pattern of neo-synthetized proteins (see text).
	Fig 4. PP242 delays cyclin B/CDK1 activation triggered by fertilization.(A) S. granularis eggs were fertilized in the absence (control) or presence of 10μM PP242 (PP242). Aliquots of total extracts from 20 μl pelleted embryos taken at indicated times post-fertilization were resolved by 12% SDS-PAGE and subjected to Western blotting analysis using phospho-PP1Cα antibody (top panels). CDK1 immunolabelling with PSTAIR antibody was used as a loading control for Western blot (bottom panels). (B) Quantitation of the results obtained (in A) from control (circles) or PP242-treated triangles) embryos. Phospho318T-PP1Cα levels were normalized against CDK1 levels obtained at the same time and expressed as a ratio of the value obtained with unfertilized eggs. (C) Quantitation of Phospho318T-PP1Cα levels obtained from untreated (white boxes) and PP242-treated (grey boxes) eggs at the indicated time following fertilization. Phospho318T-PP1Cα level was normalized against CDK1 level and expressed as a percentage of the value obtained with unfertilized eggs. Vertical bars represent Standard Error of the Mean values (SEM) obtained in 6 independent experiments. (*) Indicates a significant difference in Phospho318T-PP1Cα level in the PP242-treated embryos in comparison to the untreated embryos (Wilcoxon signed-rank test, p<0.05).
	Fig 5. PP242 delays cyclin B protein accumulation triggered by fertilization.(A) Total amount of cyclin B in extracts from control or PP242-treated embryos of S. granularis, obtained at indicated times post-fertilization was monitored by Western blotting using anti-cyclin B (top panels). CDK1 immunolabelling with PSTAIR antibody was used as a loading control for Western blot (bottom panels). (B) Quantification of the results obtained from control (circles) or PP242-treated (triangles) embryos. Cyclin B level, the sum of the two immunorevealed bands, was normalized against CDK1 level and expressed as a ratio of the value obtained with unfertilized eggs. (C) Quantitation of cyclin B level obtained from untreated (white boxes) and PP242-treated (grey boxes) embryos at the indicated time following fertilization. Cyclin B level was normalized against CDK1 level and expressed as a percentage of the value obtained with unfertilized eggs. Vertical bars represent Standard Error of the Mean values (SEM) obtained in 6 independent experiments. (*) indicates a significant difference in cyclin B level in the PP242-treated embryos in comparison to the untreated embryos (Wilcoxon signed-rank test, p<0.05).
	Fig 6. PP242 affects early embryonic development in P. lividus.(A) PP242 delays the first mitotic division triggered by fertilization. P. lividus eggs were incubated 10 min before sperm addition with DMSO (circles) or 10 μM (triangles) PP242. (B) PP242 affects the increase of protein synthesis. Embryos were metabolically labelled in the presence of [35S]methionine and the rate of in vivo protein synthesis was monitored in eggs fertilized in absence (circles) or presence of 10 μM PP242 (triangles). (C) Pattern of proteins translated following fertilization of control (left panel) or PP242-treated (right) embryos. (D) PP242 inhibits 4E-BP degradation triggered by fertilization. Total amount of 4E-BP in unfertilized eggs (UnF) or 60 min post-fertilized (F) embryos treated or not with PP242 was analysed by Western blotting using sea urchin 4E-BP antibodies (bottom panel). eIF4E immunolabelling with eIF4E antibody was used as a loading control (top panel). (E) PP242 affects CDK1/cyclin B activation triggered by fertilization. CDK1/cyclin B activation was monitored by Western blot analysis of the phospho318T-PP1Cα level. CDK1 immunolabelling with PSTAIR antibody was used as a loading control for Western blot. These figures are representative of 3 complete sets of experiments done with different females.

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